The small GTPase RhoA causes growth cone collapse and halts axon growth, thereby inhibiting regeneration of injured nerves. In contrast, RhoA has also been shown to promote growth cone advance under specific circumstances, and is important for guidance in growth cone motility. The broad objective of this proposal is to deduce how, through precise control of RhoA activation kinetics and localization, this protein can stimulate opposite cell behaviors. To resolve this conflict, we will utilize a biosensor to directly visualize the activation of RhoA in growing and retracting neurons, and manipulate microtubules to probe underlying mechanisms in RhoA signaling. Specific Aim 1 will determine the spatiotemporal dynamics of RhoA activation in growth cones during both extension and collapse, using novel image processing tools to generate a dynamic map of RhoA activation with subsecond and submicron resolution. The microtubule cytoskeleton is a master regulator of growth cone motility and guidance; it is both a major downstream target and upstream activator of RhoA. Specific Aim 2 will determine the relationship between RhoA activation and microtubule distribution and dynamics in growth cones. [unreadable] PUBLIC HEALTH RELEVANCE: In addition to nervous system regeneration failure, there are a host of severe and debilitating developmental diseases in which axon growth fails. Understanding the basic mechanism behind growth cone motility and axon extension can lead to the development of new treatments for nervous system injury and disease. [unreadable] [unreadable]